Supramolecular organization of photosystem I and light-harvesting complex I in Chlamydomonas reinhardtii

We report a structural characterization by electron microscopy and image analysis of a supramolecular complex consisting of photosystem I and light-harvesting complex I from the unicellular green alga Chlamydomonas reinhardtii. The complex is a monomer, has longest dimensions of 21.3 and 18.2 nm in projection, and is significantly larger than the corresponding complex in spinach. Comparison with photosystem I complexes from other organisms suggests that the complex contains about 14 light-harvesting proteins, two or three of which bind at the side of the PSI-H subunit. We suggest that special light-harvesting I proteins play a role in the binding of phosphorylated light-harvesting complex II in state 2.     

Chlorophyll b expressed in Cyanobacteria functions as a light-harvesting antenna in photosystem I through flexibility of the proteins

Photosynthetic pigments bind to their specific proteins to form pigment-protein complexes. To investigate the pigment-binding activities of the proteins, chlorophyll b was for introduced the first time to a cyanobacterium that did not synthesize that pigment, and expression of its function in the native pigment-protein complex of cyanobacterium was confirmed by energy transfer. Arabidopsis CAO (chlorophyll a oxygenase) cDNA was introduced into the genome of Synechocystis sp. PCC6803. The transformant cells accumulated chlorophyll b, with the chlorophyll b content being in the range of 1.4 to 10.6% of the total chlorophyll depending on the growth phase. Polyacrylamide gel electrophoresis analysis of the chlorophyll-protein complexes of transformant cells showed that chlorophyll b was incorporated preferentially into the P700-chlorophyll a-protein complex (CP1). Furthermore, chlorophyll b in CP1 transferred light energy to chlorophyll a, indicating a functional transformation. We also found that CP1 of Chlamydomonas reinhardtii, believed to be a chlorophyll a protein, bound chlorophyll b with a chlorophyll b content of approximately 4.4%. On the basis of these results, the evolution of pigment systems in an early stage of cyanobacterial development is discussed in this paper.     

Chlorophyll proteins of photosystem I

Data are presented which suggest the existence of a light-harvesting pigment-protein complex which is functionally and structurally associated with photosystem I (PSI) reaction centers. These observations are based on techniques which allow isolation of PSI using minimal concentrations of Triton X-100. Properties of density and self aggregation allowed purification of a “native” PSI complex.     

The light-harvesting complex of photosystem I in Chlamydomonas reinhardtii: protein composition, gene structures and phylogenic implications

Light-harvesting chlorophyll a/b-binding proteins (LHCI) associated with photosystem I (PSI) and the genes encoding these proteins have been characterized in the unicellular green alga Chlamydomonas reinhardtii, extending previous studies of the PSII-LHCII [Teramoto et al. (2001) Plant Cell Physiol. 42: 849]. In order to assign LHCI proteins in the thylakoid membranes, the PSI-LHCI supercomplex that retains all of the major LHCI proteins was purified. Seven distinct LHCI proteins were resolved from the purified supercomplex by a high-resolution SDS polyacrylamide gel electrophoresis, and their N-terminal amino acid sequences were determined. One LHCI protein (band e) was newly found, although the other six LHCI proteins corresponded to those previously reported. Genomic clones encoding these seven LHCI proteins were newly isolated and the nucleotide sequences were determined. A comprehensive characterization of all members of Lhc gene family in this alga revealed that LHCI proteins are more highly diverged than LHCII, suggesting functional differentiation of the protein components in LHCI. Neighbor joining trees were constructed for LHC proteins from C. reinhardtii and those of Arabidopsis thaliana or Galdieria sulphuraria to assess evolutionary relationships. Phylogenetic analysis revealed that (1). green algal LHCI and LHCII proteins are more closely related to one another than to LHCI proteins in red algae, (2). green algae and higher plants possess seven common lineages of LHC proteins, and (3). Type I and III LHCI proteins are conserved between green algae and higher plants, while Type II and IV are not. These findings are discussed in the context of evolution of multiple diverse antenna complexes.     

The PSI-K subunit of photosystem I is involved in the interaction between light-harvesting complex I and the photosystem I reaction center core

PSI-K is a subunit of photosystem I. The function of PSI-K was characterized in Arabidopsis plants transformed with a psaK cDNA in antisense orientation, and several lines without detectable PSI-K protein were identified. Plants without PSI-K have a 19% higher chlorophyll a/b ratio and 19% more P700 than wild-type plants. Thus, plants without PSI-K compensate by making more photosystem I. The photosystem I electron transport in vitro is unaffected in the absence of PSI-K. Light response curves for oxygen evolution indicated that the photosynthetic machinery of PSI-K-deficient plants have less capacity to utilize light energy. Plants without PSI-K have less state 1-state 2 transition. Thus, the redistribution of absorbed excitation energy between the two photosystems is reduced. Low temperature fluorescence emission spectra revealed a 2-nm blue shift in the long wavelength emission in plants lacking PSI-K. Furthermore, thylakoids and isolated PSI without PSI-K had 20-30% less Lhca2 and 30-40% less Lhca3, whereas Lhca1 and Lhca4 were unaffected. During electrophoresis under mildly denaturing conditions, all four Lhca subunits were partially dissociated from photosystem I lacking PSI-K. The observed effects demonstrate that PSI-K has a role in organizing the peripheral light-harvesting complexes on the core antenna of photosystem I.     

Pigment binding of photosystem I light-harvesting proteins

Light-harvesting complexes (LHC) of higher plants are composed of at least 10 different proteins. Despite their pronounced amino acid sequence homology, the LHC of photosystem II show differences in pigment binding that are interpreted in terms of partly different functions. By contrast, there is only scarce knowledge about the pigment composition of LHC of photosystem I, and consequently no concept of potentially different functions of the various LHCI exists. For better insight into this issue, we isolated native LHCI-730 and LHCI-680. Pigment analyses revealed that LHCI-730 binds more chlorophyll and violaxanthin than LHCI-680. For the first time all LHCI complexes are now available in their recombinant form; their analysis allowed further dissection of pigment binding by individual LHCI proteins and analysis of pigment requirements for LHCI formation. By these different approaches a correlation between the requirement of a single chlorophyll species for LHC formation and the chlorophyll a/b ratio of LHCs could be detected, and indications regarding occupation of carotenoid-binding sites were obtained. Additionally the reconstitution approach allowed assignment of spectral features observed in native LHCI-680 to its components Lhca2 and Lhca3. It is suggested that excitation energy migrates from chlorophyll(s) fluorescing at 680 (Lhca3) via those fluorescing at 686/702 nm (Lhca2) or 720 nm (Lhca3) to the photosystem I core chlorophylls.     

Chlorophyll synthesis modulates retention of apoproteins of light-harvesting complex II by the chloroplast in Chlamydomonas reinhardtii

Localization of apoproteins of the major light-harvesting complex (LHCII) in Chl b -less cells of Chlamydomonas reinhardtii cbn 1–113 was determined by immunoelectron microscopy. In dark-grown cells, a low amount of apoproteins was detected in cytoplasmic vacuoles. The amount in vacuoles, and in the cytosol, increased dramatically when the rate of protein synthesis was enhanced in the dark by raising the temperature to 38°C. After exposure of cells to light, the apoproteins accumulated also in the chloroplast. Mature-sized apoproteins were recovered in an alkali-soluble fraction of cellular proteins commensurate with accumulation in the cytoplasm. At 25°C, content of apoproteins in the chloroplast of pale-green cells grown in medium lacking acetate was one-half of the amount in cells grown with acetate, yet the total amount remained similar. Cytoplasmic vacuoles, which were nearly filled with immunoreactive, electron-opaque material, were more abundant in cells grown without acetate as compared with cells grown with acetate. Accumulation of apoproteins outside of the chloroplast suggested that translocation into the organelle of a portion of the apoproteins, apparently synthesized in excess of the amount accommodated by Chl synthesis, was aborted after processing of precursors. These results suggested that assembly of LHCII was required for retention of apoproteins by the chloroplast.     

PSI-O, a new 10-kDa subunit of eukaryotic photosystem I

A novel polypeptide with an apparent molecular mass of 9 kDa was detected after sodium dodecyl sulphate–polyacrylamide gel electrophoresis of Arabidopsis photosystem I (PSI) and was N-terminally sequenced. Corresponding cDNA clones encode a precursor protein of 140 amino acid residues which was imported into isolated intact chloroplasts and processed to the mature protein, designated PSI-O. The mature protein has two transmembrane helices and a calculated mass of 10 104 Da. The PSI-O protein was also shown to be present in PSI isolated from barley and spinach, and was essentially absent in chloroplast grana. Expressed sequences encoding similar proteins are available from many species of plants and green algae.     

Green plant photosystem I binds light-harvesting complex I on one side of the complex

We report a structural characterization by electron microscopy of green plant photosystem I solubilized by the mild detergent n-dodecyl-alpha-D-maltoside. It is shown by immunoblotting that the isolated complexes contain all photosystem I core proteins and all peripheral light-harvesting proteins. The electron microscopic analysis is based on a large data set of 14 000 negatively stained single-particle projections and reveals that most of the complexes are oval-shaped monomers. The monomers have a tendency to associate into artificial dimers, trimers, and tetramers in which the monomers are oppositely oriented. Classification of the dimeric complexes suggests that some of the monomers lack a part of the peripheral antenna. On the basis of a comparison with projections from trimeric photosystem I complexes from cyanobacteria, we conclude that light-harvesting complex I only binds to the core complex at the side of the photosystem I F/J subunits and does not cause structural hindrances for the type of trimerization observed in cyanobacterial photosystem I.     

Molecular design of the photosystem II light-harvesting antenna: photosynthesis and photoprotection

The photosystem II (PSII) light-harvesting system carries out two essential functions, the efficient collection of light energy for photosynthesis, and the regulated dissipation of excitation energy in excess of that which can be used. This dual function requires structural and functional flexibility, in which light-harvesting proteins respond to an external signal, the thylakoid DeltapH, to induce feedback control. This process, referred to as non-photochemical quenching (NPQ) depends upon the xanthophyll cycle and the PsbS protein. In nature, NPQ is heterogeneous in terms of kinetics and capacity, and this adapts photosynthetic systems to the specific dynamic features of the light environment. The molecular features of the thylakoid membrane which may enable this flexibility and plasticity are discussed.     

Congenital deficiency of a 20-kDa subunit of mitochondrial complex I in fibroblasts.

The first component of the mitochondrial electron-transport chain is especially complex, consisting of 19 nuclear and seven mitochondrion-encoded subunits. Accordingly, a wide range of clinical manifestations are produced by the various mutations occurring in human populations. In this study, we analyze the subunit structure of complex I in fibroblasts from two patients who have distinct clinical phenotypes associated with complex I deficiency. The first patient died in the second week of life from overwhelming lactic acidosis. Severe complex I deficiency was evident in her fibroblasts, since alanine oxidation was markedly reduced whereas succinate oxidation was normal. Absence of a 20-kDa subunit was demonstrable when newly synthesized proteins were immunoprecipitated from pulse-labeled fibroblasts by anti-complex I antibody. Disordered assembly or decreased stability of the complex was suggested by deficiency of multiple subunits on Western immunoblots. The second patient exhibited a milder clinical phenotype, characterized by moderate lactic acidosis and developmental delay in childhood and by onset of seizures at 8 years of age. Oxidation studies demonstrated expression of the complex I deficiency in fibroblasts, but no subunit abnormalities were detected by immunoprecipitation or Western immunoblotting. This report demonstrates the utility of cultured fibroblasts in studying mutations affecting synthesis and assembly of complex I.     

Proteomic analysis of the photosystem I light-harvesting antenna in tomato (Lycopersicon esculentum)

Until now, more genes of the light-harvesting antenna of higher-plant photosystem I (PSI) than proteins have been described. To improve our understanding of the composition of light-harvesting complex I (LHCI) of tomato (Lycopersicon esculentum), we combined one- and two-dimensional (1-D and 2-D, respectively) gel electrophoresis with immunoblotting and tandem mass spectrometry (MS/MS). Separation of PSI with high-resolution 1-D gels allowed separation of five bands attributed to proteins of LHCI. Immunoblotting with monospecific antibodies and MS/MS analysis enabled the correct assignment of the four prominent bands to light-harvesting proteins Lhca1-4. The fifth band was recognized by only the Lhca1 antibody. Immunodetection as well as mass spectrometric analysis revealed that these protein bands contain not only the eponymous protein but also other Lhca proteins, indicating a heterogeneous protein composition of Lhca bands. Additionally, highly sensitive MS/MS allowed detection of a second Lhca4 isoform and of Lhca5. These proteins had not been described before on the protein level in higher plants. Two-dimensional gel electrophoresis revealed an even more diverse composition of individual Lhca proteins than was apparent from 1-D gels. For each of the four prominent Lhca proteins, four to five isoforms with different isoelectric points could be identified. In the case of Lhca1, Lhca4, and Lhca3, additional isoforms with slightly differing molecular masses were identified. Thus, we were able to detect four to ten isoforms of each individual Lhca protein in PSI. Reasons for the origin of Lhca heterogeneity are discussed. The observed variety of Lhca proteins and their isoforms is of particular interest in the context of the recently published crystal structure of photosystem I from pea, which showed the presence of only four Lhca proteins per photosystem I. These findings indicate that several populations of photosystem I that differ in their Lhca composition may exist.     

Fluorescence emission spectra of photosystem I, photosystem II and the light-harvesting chlorophyll a/b complex of higher plants

Fluorescence emission spectra excited at 514 and 633 nm were measured at -196 degrees C on dark-grown bean leaves which had been partially greened by a repetitive series of brief xenon flashes. Excitation at 514 nm resulted in a greater relative enrichment of the 730 nm emission band of Photosystem I than was obtained with 633 nm excitation. The difference spectrum between the 514 nm excited fluorescence and the 633 nm excited fluorescence was taken to be representative of a pure Photosystem I emission spectrum at -196 degrees C. It was estimated from an extrapolation of low temperature emission spectra taken from a series of flashed leaves of different chlorophyll content that the emission from Photosystem II at 730 nm was 12% of the peak emission at 694 nm. Using this estimate, the pure Photosystem I emission spectrum was subtracted from the measured emission spectrum of a flashed leaf to give an emission spectrum representative of pure Photosystem II fluorescence at -196 degrees C. Emission spectra were also measured on flashed leaves which had been illuminated for several hours in continuous light. Appreciable amounts of the light-harvesting chlorophyll a/b protein, which has a low temperature fluorescence emission maximum at 682 nm, accumulate during greening in continuous light. The emission spectra of Photosystem I and Photosystem II were subtracted from the measured emission spectrum of such a leaf to obtain the emission spectrum of the light-harvesting chlorophyll a/b protein at -196 degrees C.     

De-epoxidation of violaxanthin in light-harvesting complex I proteins

The conversion of violaxanthin (Vx) to zeaxanthin (Zx) in the de-epoxidation reaction of the xanthophyll cycle plays an important role in the protection of chloroplasts against photooxidative damage. Vx is bound to the antenna proteins of both photosystems. In photosystem II, the formation of Zx is essential for the pH-dependent dissipation of excess light energy as heat. The function of Zx in photosystem I is still unclear. In this work we investigated the de-epoxidation characteristics of light-harvesting complex proteins of photosystem I (LHCI) under in vivo and in vitro conditions. Recombinant LHCI (Lhcal-4) proteins were reconstituted with Vx and lutein, and the convertibility of Vx was studied in an in vitro assay using partially purified Vx de-epoxidase isolated from spinach thylakoids. All four LHCI proteins exhibited unique de-epoxidation characteristics. An almost complete Vx conversion to Zx was observed only in Lhca3, whereas Zx formation in the other LHCI proteins decreased in the order Lhca4 > Lhca1 > Lhca2. Most likely, these differences in Vx de-epoxidation were related to the different accessibility of the respective carotenoid binding sites in the distinct antenna proteins. The results indicate that Vx bound to site V1 and N1 is easily accessible for de-epoxidation, whereas Vx bound to L2 is only partially and/or with the slower kinetics convertible to Zx. The de-epoxidation properties determined for the monomeric recombinant proteins were consistent with those obtained for isolated native LHCI-730 and LHCI-680 in the same in vitro assay and the de-epoxidation state found under in vivo conditions in native LHCIs.     

Structural characterization of a complex of photosystem I and light-harvesting complex II of Arabidopsis thaliana

Chloroplasts are central to the provision of energy for green plants. Their photosynthetic membrane consists of two major complexes converting sunlight: photosystem I (PSI) and photosystem II (PSII). The energy flow toward both photosystems is regulated by light-harvesting complex II (LHCII), which after phosphorylation can move from PSII to PSI in the so-called state 1 to state 2 transition and can move back to PSII after dephosphorylation. To investigate the changes of PSI and PSII during state transitions, we studied the structures and frequencies of all major membrane complexes from Arabidopsis thaliana chloroplasts at conditions favoring either state 1 or state 2. We solubilized thylakoid membranes with digitonin and analyzed the complete set of complexes immediately after solubilization by electron microscopy and image analysis. Classification indicated the presence of a PSI-LHCII supercomplex consisting of one PSI-LHCI complex and one LHCII trimer, which was more abundant in state 2 conditions. The presence of LHCII was confirmed by excitation spectra of the PSI emission of membranes in state 1 or state 2. The PSI-LHCII complex could be averaged with a resolution of 16 A, showing that LHCII has a specific binding site at the PSI-A, -H, -L, and -K subunits.     

Quenching of chlorophyll triplet states by carotenoids in reconstituted Lhca4 subunit of peripheral light-harvesting complex of photosystem I

In this study, triplet quenching, the major photoprotection mechanism in antenna proteins, has been studied in the light-harvesting complex of photosystem I (LHC-I). The ability of carotenoids bound to LHC-I subunit Lhca4, which is characterized by the presence of the red-most absorption components at wavelength >700 nm, to protect the system through quenching of the chlorophyll triplet states, has been probed, by analyzing the induction of carotenoid triplet formation. We have investigated this process at low temperature, when the funneling of the excitation toward the low-lying excited states of the Chls is stronger, by means of optically detected magnetic resonance (ODMR), which is well-suited for investigation of triplet states in photosynthetic systems. The high selectivity and sensitivity of the technique has made it possible to point out the presence of specific interactions between carotenoids forming the triplet states and specific chlorophylls characterized by red-shifted absorption, by detection of the microwave-induced Triplet minus Singlet (T-S) spectra. The effect of the red forms on the efficiency of triplet quenching was specifically probed by using the Asn47His mutant, in which the red forms have been selectively abolished (Morosinotto, T., Breton, J., Bassi, R., and Croce, R. (2003) J. Biol. Chem. 278, 49223-49229). Lack of the red forms yields into a reduced efficiency of the triplet quenching in LHC-I thus suggesting that the "red Chls" play a role in enhancing triplet quenching in LHC-I and, possibly, in the whole photosystem I.     

A chlorophyll-protein complex lacking in photosystem I mutants of Chlamydomonas reinhardtii

Sodium dodecyl sulfate gel electrophoresis of unheated, detergent-solubilized thylakoid membranes of Chlamydomonas reinhardtii gives two chlorophyll-protein complexes. Chlorophyll-protein complex I (CP I) is the blue-green in color and can be dissociated by heat into "free" chlorophyll and a constituent polypeptide (polypeptide 2; mol wt 66,000). Similar experiments with spinach and Chinese cabbage show that the higher plant CP I contains an equivalent polypeptide but of slightly lower molecular weight (64,000). Both polypeptide 2 and its counterpart in spinach are soluble in a 2:1 (vol/vol) mixture of chloroform-methanol. Chemical analysis reveals that C. reinhardtii CP I has a chlorophyll a to b weight ratio of about 5 and that it contains approximately 5% of the total chlorophyll and 8-9% of the total protein of the thylakoid membranes. Thus, it can be calculated that each constituent polypeptide chain is associated with eight to nine chlorophyll molecules. Attempts to measure the molecular weight of CP I by calibrated SDS gels were unsuccessul since the complex migrates anomalously in such gels. Two Mendelian mutants of C. reinhardtii, F1 and F14, which lack P700 but have normal photosystem I activity, do not contain CP I or the 66,000-dalton polypeptide in their thylakoid membranes. Our results suggest that CP I is essential for photosystem I reaction center activity and that P700 may be associated with the 66,000-dalton polypeptide.     

Supercomplexes of plant photosystem I with cytochrome b6f,light-harvesting complex II and NDH

Photosystem I (PSI) is a pigment-protein complex required for the light-dependent reactions of photosynthesis and participates in light-harvesting and redox-driven chloroplast metabolism. Assembly of PSI into supercomplexes with light harvesting complex (LHC) II, cytochrome b₆f (Cytb₆f) or NAD(P)H dehydrogenase complex (NDH) has been proposed as a means for regulating photosynthesis. However, structural details about the binding positions in plant PSI are lacking. We analyzed large data sets of electron microscopy single particle projections of supercomplexes obtained from the stroma membrane of Arabidopsis thaliana. By single particle analysis, we established the binding position of Cytb₆f at the antenna side of PSI. The rectangular-shaped Cytb₆f dimer binds at the side where Lhca1 is located. The complex binds with its short side rather than its long side to PSI, which may explain why these supercomplexes are difficult to purify and easily disrupted. Refined analysis of the interaction between PSI and the NDH complex indicates that in total up to 6 copies of PSI can arrange with one NDH complex. Most PSI-NDH supercomplexes appeared to have 1–3 PSI copies associated. Finally, the PSI-LHCII supercomplex was found to bind an additional LHCII trimer at two positions on the LHCI side in Arabidopsis. The organization of PSI, either in a complex with NDH or with Cytb₆f, may improve regulation of electron transport by the control of binding partners and distances in small domains.